生物活性

Inhibitor of Wnt processing and secretion. Inactivates PORCN, a membrane-bound O-acyltransferase (MBOAT), and selectively inhibits palmitoylation of Wnt. Blocks Wnt-dependent phosphorylation of Lrp6 receptor and Dvl2, and β-catenin accumulation. Suppresses self-renewal in R1 embryonic stem cells. 

Wnt pathway activity: IC50= 27 nM[1]

体外研究

体内研究

激酶实验

细胞实验

Soft agar assay[2]

Stably transduced AsPC-1 or HPAF-2 cells (5000 or 15 000 cells per well of24-well plate, respectively) were plated as single cell suspensions in 0.4% Noble Agar overlaid on a base layer of 0.8% Noble Agar. Media (RPMI-1640+10% fetal bovine serum) was replaced every 3 days. After 2–3 weeks, colonies were visualized by incubation with 10% MTT reagent for5 h. Colonies greater than 200 mm in diameter were counted.

Sphere assay

Indicated HPAF-2 cells were suspended in Neurobasal Media containing Glutamax, bFGF (20ng/ml), EGF (20ng/ml), N-2 and B27 at 10 000 cells/well in 6-well ultra-low attachment plates. After 8 days, tumor spheres were visualized and counted by phase contrast microscopy.

动物实验

Mice Infection[3]

About 3-mo-old C57BL/6 mice were obtained and housed four to five in a cage at 23 °C in a 12-h light/dark cycle. Mice were injected intraperitoneally (i.p.) first with either 200 μL of liposome-IWP2 (LI) or liposome (L) and then after 2 h with 1 × 108 or 2 × 108 CFU E. coli in 200 μL of sterile PBS. After 2 h or 24 h mice were killed, and the peritoneal cavity was washed with 5 mL of sterile ice-cold PBS. The peritoneal lavage fluid was centrifuged at 300 × g for 5 min, the cell pellet was resuspended in RPMI 1640 complete medium, and the supernatant was used for cytokine assay. For ex vivo experiments, peritoneal phagocytes were isolated as above from normal mice, and equal numbers of cells were plated in medium overnight at 37 °C in 5% CO2before performing further experiments.

Estimation of Phagocytosis

Latex beads, either blue dye filled (0.8μm) or fluorescent red (2μm), were added to transfected RAW cells (about 10 beads per cell), about 60 h after transfection, and the plates were incubated at 37 °C in 5% CO2for 6 h. Extracellular particles were removed by washing the cells extensively with icecold PBS several times. In the case of fluorescent beads, cells were treated for 1 min with trypan blue after the required incubation to quench extracellular fluorescence. Cells were subsequently harvested for assay. Fluorescence trapped by cells was measured by fluorometer using excitation and emission wavelengths of 575 nm and 610 nm, respectively. Uptake of blue-dye-filled beads was estimated by measuring absorbance at 595 nm. Uptake of red fluorescent beads by RAW cells was assessed separately by confocal microscopy. To assess uptake of GFP expressing E. coli [E. coli DH5α bacteria (avirulent) transformed with Green Fluorescent Protein plasmid], RAW cells pretreated with L-Wnt5a or L-Wnt3a conditioned medium (150 μL in 1 mL of culture medium) or recombinant Wnt5a (50 ng/mL, dissolved in PBS with 0.1%BSA) for 6 h were incubated with GFP–E. coli at a multiplicity of infection (MOI) of 50 for 120 min at 37 °C in 5% CO2. Unbound bacteria were removed by extensive washing several times with cold PBS. Infected RAW cells were subsequently lysed by adding distilled water. The diluted aliquots were then spread on LB agar plate and colony -forming units (CFUs) were counted after incubating the plates overnight at 37 °C. As control experiments, PBS with 0.1% BSA was used instead of recombinant Wnt5a. Both peritoneal macrophages and RAW cells pretreated with either 0.05 μM IWP-2 or the liposome formulation for 48 h was processed similarly to assess phagocytosis. For estimating inhibition by designated inhibitors, specific concentrations as noted in the figure legends were added for the last 2 h of Wnt5a or PBS incubation before addition of GFP–E. coli or latex bead.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Chen B, Dodge ME, Tang W, et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat Chem Biol. 2009;5(2):100-107.
[2] Arensman MD, Kovochich AN, Kulikauskas RM, et al. WNT7B mediates autocrine Wnt/beta-catenin signaling and anchorage-independent growth in pancreatic adenocarcinoma. Oncogene. 2014;33(7):899-908.
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体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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